ABCMdb

PMID: 21685390

Marquer C, Fruchart-Gaillard C, Letellier G, Marcon E, Mourier G, Zinn-Justin S, Menez A, Servent D, Gilquin B
Structural model of ligand-G protein-coupled receptor (GPCR) complex based on experimental double mutant cycle data: MT7 snake toxin bound to dimeric hM1 muscarinic receptor.
J Biol Chem. 2011 Sep 9;286(36):31661-75. Epub 2011 Jun 17., [PubMed]

Sentences

No. Mutations Sentence Comment

156 ABCC8 p.Leu183Ala ABCC8 p.Tyr179Phe hM1 mutant Region pKi NMS pKX MT7 KX(mut)/KX(WT) WT 10.22 Ϯ 0.04 10.47 Ϯ 0.04 H90A E1 10.20 Ϯ 0.09 10.66 Ϯ 0.20 0.6 W91A E1 9.23 ؎ 0.33 9.30 ؎ 0.06 14.9 W101A TM3 9.29 Ϯ 0.02 9.79 Ϯ 0.13 4.8 Y166A E2 9.83 Ϯ 0.03 10.33 Ϯ 0.32 1.4 L167F E2 9.93 Ϯ 0.02 10.48 Ϯ 0.11 1.0 E170A E2 10.20 Ϯ 0.04 10.20 Ϯ 0.07 1.9 E170K E2 10.16 Ϯ 0.02 9.31 ؎ 0.11 14.6 R171A E2 9.76 Ϯ 0.01 9.50 ؎ 0.29 9.4 L174A E2 10.21 Ϯ 0.08 10.51 Ϯ 0.01 0.9 L174P E2 10.15 Ϯ 0.01 9.37 ؎ 0.14 12.5 Q177E E2 10.08 Ϯ 0.04 10.58 Ϯ 0.17 0.8 Y179F E2 9.85 Ϯ 0.02 9.25 ؎ 0.18 16.5 L183A E2 10.00 Ϯ 0.11 10.44 Ϯ 0.20 1.1 Q185A E2 10.18 Ϯ 0.10 10.59 Ϯ 0.37 0.8 K392A E3 10.20 Ϯ 0.12 10.57 Ϯ 0.18 0.8 D393A E3 9.96 Ϯ 0.09 10.50 Ϯ 0.23 0.9 E397A E3 10.21 Ϯ 0.06 10.54 Ϯ 0.20 0.9 W400A TM7 10.12 Ϯ 0.04 9.43 ؎ 0.02 11.0 E401A TM7 9.76 Ϯ 0.02 10.20 Ϯ 0.10 1.9 TABLE 2 Affinity constants and variations in free energy of interaction of wild-type and modified MT7 toxins for wild-type and mutated hM1 receptors Data were inferred from binding experiments with ͓3 H͔NMS and calculated with the ternary complex equation. Login to comment 160 ABCC8 p.Tyr179Phe hM1 receptor hM1 region MT7 toxin MT7 loop pKX ⌬⌬Gint kcal/mol WT WT 10.47 Ϯ 0.04 H90A E1 WT 10.66 Ϯ 0.20 W10A 1 11.62 Ϯ 0.28 -2.08 Ϯ 0.40 Y30A 2 9.55 Ϯ 0.28 ؉1.14 Ϯ 0.37 Y51A 3 10.42 Ϯ 0.13 ϩ0.20 Ϯ 0.11 R52A 3 10.42 Ϯ 0.44 -0.04 Ϯ 0.59 W91A E1 WT 9.30 Ϯ 0.06 W10A 1 7.69 Ϯ 0.05 ؉1.09 Ϯ 0.07 Y30A 2 7.81 Ϯ 0.07 ؉1.64 Ϯ 0.09 Y51A 3 8.79 Ϯ 0.17 ϩ0.44 Ϯ 0.24 R52A 3 8.54 Ϯ 0.13 ϩ0.66 Ϯ 0.17 W101A TM3 WT 9.79 Ϯ 0.13 W10A 1 8.57 Ϯ 0.14 ϩ0.57 Ϯ 0.19 Y30A 2 9.08 Ϯ 0.09 ϩ0.60 Ϯ 0.11 R34A 2 7.74 Ϯ 0.10 -0.37 Ϯ 0.21 R52A 3 9.05 Ϯ 0.19 ϩ0.62 Ϯ 0.25 E170A E2 WT 10.20 Ϯ 0.07 W10A 1 8.42 Ϯ 0.20 ؉1.33 Ϯ 0.25 Y30A 2 8.84 Ϯ 0.28 ؉1.47 Ϯ 0.38 S32A 2 8.63 Ϯ 0.16 ؉1.43 Ϯ 0.22 R34A 2 7.50 Ϯ 0.36 ϩ0.60 Ϯ 0.49 M35A 2 8.63 Ϯ 0.13 ؉0.87 Ϯ 0.17 Y36A 2 7.97 Ϯ 0.13 ؉1.24 Ϯ 0.17 Y51A 3 9.71 Ϯ 0.31 ϩ0.41 Ϯ 0.42 R52A 3 8.57 Ϯ 0.18 ؉1.77 Ϯ 0.27 R171A E2 WT 9.50 Ϯ 0.29 K5A 1 9.25 Ϯ 0.18 -0.10 Ϯ 0.24 W10A 1 7.76 Ϯ 0.18 ؉1.26 Ϯ 0.24 Y30A 2 8.22 Ϯ 0.14 ؉1.49 Ϯ 0.43 S32A 2 8.46 Ϯ 0.08 ؉0.72 Ϯ 0.11 M35A 2 7.68 Ϯ 0.46 ؉1.20 Ϯ 0.62 Y36A 2 7.03 Ϯ 0.21 ؉1.55 Ϯ 0.28 K48A 2 9.48 Ϯ 0.02 -0.13 Ϯ 0.02 Y51A 3 8.67 Ϯ 0.12 ؉0.89 Ϯ 0.16 R52A 3 8.35 Ϯ 0.17 ؉1.17 Ϯ 0.23 L174A E2 WT 10.51 Ϯ 0.01 W10A 1 9.19 Ϯ 0.20 ؉0.71 ؎ 0.27 Y30A 2 9.45 Ϯ 0.17 ؉1.08 Ϯ 0.23 S32A 2 9.66 Ϯ 0.20 ϩ0.48 Ϯ 0.27 R34A 2 7.52 Ϯ 0.13 ؉1.19 Ϯ 0.42 M35A 2 8.72 Ϯ 0.10 ؉1.16 Ϯ 0.13 Y36A 2 8.59 Ϯ 0.23 ؉0.83 Ϯ 0.31 Y51A 3 10.06 Ϯ 0.23 ϩ0.37 Ϯ 0.31 R52A 3 9.23 Ϯ 0.22 ؉1.35 Ϯ 0.29 Y179F E2 WT 9.25 Ϯ 0.18 K5A 1 9.15 Ϯ 0.15 -0.28 Ϯ 0.20 W10A 1 7.62 Ϯ 0.19 ؉1.13 Ϯ 0.25 R34A 2 Ͻ6 > ؉1.35 K48A 2 9.21 Ϯ 0.01 -0.09 Ϯ 0.01 R52A 3 Ͻ6 >؉4.00 K392A E3 WT 10.57 Ϯ 0.18 W10A 1 9.88 Ϯ 0.22 -0.13 Ϯ 0.29 F11A 1 10.19 Ϯ 0.20 ϩ0.27 Ϯ 0.27 D393A E3 WT 10.50 Ϯ 0.23 S8A 1 10.05 Ϯ 0.06 ϩ0.21 Ϯ 0.08 W10A 1 10.10 Ϯ 0.02 -0.53 Ϯ 0.02 F11A 1 10.21 Ϯ 0.24 ϩ0.35 Ϯ 0.09 Y51A 3 10.00 Ϯ 0.34 ϩ0.42 Ϯ 0.46 E397A E3 WT 10.54 Ϯ 0.20 W10A 1 8.64 Ϯ 0.22 ؉1.58 Ϯ 0.31 F11A 1 9.89 Ϯ 0.21 ϩ0.64 Ϯ 0.28 W400A TM7 WT 9.43 Ϯ 0.02 K5A 1 9.60 Ϯ 0.05 -0.66 Ϯ 0.07 W10A 1 7.61 Ϯ 0.32 ؉1.38 Ϯ 0.44 F11A 1 8.29 Ϯ 0.25 ؉1.29 Ϯ 0.33 Y30A 2 8.49 Ϯ 0.08 ؉0.90 Ϯ 0.10 S32A 2 8.64 Ϯ 0.15 ϩ0.38 Ϯ 0.20 R34A 2 Ͻ6 >؉1.58 M35A 2 8.52 Ϯ 0.13 -0.11 Ϯ 0.12 Y36A 2 7.37 Ϯ 0.29 ؉1.00 Ϯ 0.38 K48A 3 9.41 Ϯ 0.06 -0.12 Ϯ 0.07 R52A 3 7.73 Ϯ 0.08 ؉0.91 Ϯ 0.17 E401A TM7 WT 10.20 Ϯ 0.10 R34A 2 7.41 Ϯ 0.37 ϩ0.61 Ϯ 0.45 Modeling of MT7-Dimeric hM1 Muscarinic Receptor Complex 31666 account this flexibility, we generated open conformations of loop E2 by molecular dynamics. Login to comment 271 ABCC8 p.Tyr179Trp This distance is similar to the distance between the two MT7 binding sites centered on Tyr-179/Trp-400 of each protomer in our model of MT7-hM1 dimer complex (Fig. 8B). Login to comment 277 ABCC8 p.Tyr179Trp In this case, the distance between the two MT7 binding sites (Tyr-179/Trp-400) is too large (35 Å) to receive the tips of MT7 loops (Fig. 8C). Login to comment 280 ABCC8 p.Tyr179Trp Fig. 8D shows the variation of the distances between the MT7 binding sites (Tyr-179/Trp-400) of both protomers as a function of the angle of rotation of each protomer around their vertical axis. Login to comment 283 ABCC8 p.Tyr179Trp This is due to the fact that Tyr-179/Trp- FIGURE 5. Login to comment 336 ABCC8 p.Tyr179Trp Therefore, the MT7-hM1 dimer recognition site is composed of three patches on MT7 (the tips of the three loops) and two patches on the hM1 side (the binding sites of the two protomers; Tyr-179/Trp-400), forming a totalinterfaceareaequalto2400Å2 .Thisvalueisclosetothemean valueoftheprotein/proteininterfaceareasreportedfortwo-patch interactions (2500 Å2 ) (73). Login to comment 350 ABCC8 p.Tyr179Trp B, hM1 dimer conformation calculated from our experimental data viewed from the extracellular face (Tyr-179/Trp-400 in green). Login to comment 351 ABCC8 p.Tyr179Trp C, hM1 dimer conformation based on the CXCR4 chemokine structure viewed from the extracellular face (Tyr-179/Trp-400 in yellow). Login to comment 352 ABCC8 p.Tyr179Trp D, measurements of the distancesbetweenthetwocentersofgravityoftheprotomersofthereceptor (E) and the two centers of mass of the MT7 binding sites (Tyr-179/Trp-400) of the protomers (f) during the step by step rotation of each protomer around their vertical axis. Login to comment